The present invention is generally in the field of methods for making 2-hydroxyacid monomers, and the resulting polyhydroxyalkanoate polymers.
Numerous microorganisms have the ability to accumulate intracellular reserves of PHA polymers. Poly [(R)-3-hydroxyalkanoates] (PHAs) are biodegradable thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). Around 100 different monomers have been incorporated into PHA polymers, as reported in the literature (Steinbüchel and Valentin, 1995, FEMS Microbiol. Lett. 128; 219-228) and the biology and genetics of their metabolism has recently been reviewed (Huisman and Madison, 1998, Microbiology and Molecular Biology Reviews, 63: 21-53).
To date, PHAs have seen limited commercial availability, with only the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) being available in development quantities. This copolymer has been produced by fermentation of the bacterium Ralstonia eutropha. Fermentation and recovery processes for other PHA types have also been developed using a range of bacteria including Azotobacter, Alcaligenes latus, Comamonas testosterone and genetically engineered E. coli and Klebsiella and have recently been reviewed (Braunegg et al., 1998, Journal of Biotechnology 65: 127-161; Choi and Lee, 1999, Appl. Microbiol. Biotechnol. 51: 13-21). More traditional polymer synthesis approaches have also been examined, including direct condensation and ring-opening polymerization of the corresponding lactones (Jesudason and Marchessault, 1994, Macromolecules 27: 2595-2602).
Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate (PHB4HB, Doi, Y. 1995, Macromol. Symp. 98, 585-599) or 4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters have been described (Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36, 507-514 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40, 710-716). These polyesters have been manufactured using methods similar to that originally described for PHBV in which the microorganisms are fed a relatively expensive non-carbohydrate feedstock in order to force the incorporation of the monomer into the PHA polyester. The PHB4HB copolymers can be produced with a range of monomer compositions which again provides a range of polymer (Saito, Y, Nakamura, S., Hiramitsu, M. and Doi, Y., 1996, Polym. Int. 39: 169).
PHA copolymers containing 3-hydroxyvalerate (3HV), especially poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), has been available commercially under the trade name Biopol™. PHBV has been produced commercially using Ralstonia eutropha (formerly Alcaligenes eutrophus) from carbohydrate feedstocks such as glucose in combination with a co-feed such as propionate, isobutyrate (Holmes et al., U.S. Pat. No. 4,477,654) or odd chain length alcohols or fatty acids. A number of other microorganisms and processes are known to those skilled in the art (Braunegg et al. 1998, Journal of Biotechnology 65: 127-161). PHAs containing 3HV units have also been synthesized using recombinant microorganisms. Escherichia coli harboring the R. eutropha PHA biosynthesis genes has been used to produce PHBV from glucose and either propionate or valerate (Slater et al., 1992, Appl. Environ. Microbiol. 58:1089-1094). Klebsiella oxytoca harboring the R. eutropha PHA biosynthesis genes has been used to produce PHBV from glucose and propionate (Zhang et al., 1994, Appl. Environ. Microbiol. 60:1198-1205). R. eutropha harboring the PHA synthase gene of Aeromonas caviae was used to produce poly(3HV-co-3HB-co-3HHp) from alkanoic acids of odd carbon numbers (Fukui et al., 1997, Biotechnol. Lett. 19:1093-1097). U.S. Pat. No. 6,329,183, to Skraly and Peoples, describes methods for producing PHA copolymers comprising 3HV units from 1,2-propanediol. PCT WO 00/43523 to Huisman et al., describes method for producing PHAs comprising 3-hydroxyhexanoate (3HH) monomer units from butyrate or butanol co-feeds. In each of these cases, the alcohol co-feed was converted into the free acid which was then activated to the Co-enzyme A thioester by the action of a fatty acyl-coenzymeA synthetase or fatty acyl-CoA transferase. In some cases the enzyme activity was endogenous to the host strain and in others this activity was provided by genetic engineering.
Genes and techniques for developing recombinant PHA producers suitable for practicing the disclosed invention are generally known to those skilled in the art (Madison and Huisman, 1999, Microbiology and Molecular Biology Reviews, 63: 21-53; PCT WO 99/14313).
3HV copolymers have proven useful in a range of applications. In some cases PHBV copolymers with a 3HV level of around 7-12% by weight co-monomer are adequate. In other cases a 3HV level of 15-30% by weight is more useful (EP LATEX). Higher levels of 3HV are accomplished by increasing the level of propionic acid in the feed. However, there are two negative consequences associated with this strategy. First, propionic acid is toxic to the cells and, therefore, reduces the rate of growth and polymer production representing a significant increase in the cost of production. The second effect is that some of the propionic acid can be used for other metabolic processes and is therefore not incorporated into the polymer. As the propionic acid is the most expensive of the feed components, this represents another increase in the cost of production. Therefore, it would be desirable to develop microbial systems that produce 3HV copolymers with higher productivities and better yields on the co-feed.
It is therefore an object of the present invention to provide methods and microbial strains suitable for producing PHA polymers or copolymers that avoids increasing the level of 3-hydroxyacid in the feed.
It is a further object of the present invention to provide methods and microbial strains suitable for production of PHA polymers containing 3HV units that avoids the use of 3-propionic acid in the feed.